In 1693, contagious bovine pleuropneumonia, a fetal disease of cattle, was discovered in Germany, later reaching the United States in 1843. Louis Pasteur suggested that the cause was a specific ultramicroscopic agent, since bacteria could not be detected in serous exudate capable of producing the disease in cattle. Colonies produced by these agents were difficult to detect due to their small size and inability to absorb most stains. The organisms were simply too small to be defined morphologically at that time.
A number of similar microorganisms were later isolated from animals (including humans) and sewage between 1920 and 1940. These organisms were termed pleuropneumonia-like organisms (PPLO), and are now known as mycoplasmas. As obligate cellular parasites, mycoplasmas lack the rigid peptidoglycan cell wall of eubacteria, yet contain the machinery necessary for replication in a cell-free environment.
Over 100 mycoplasma species have been identified, including more than 11 species capable of affecting humans; some being the etiologic agents of disease. Mycoplasma species include animal, plant, and insect pathogens, in addition to a significant part of the normal microbial flora of most animals. One species, Mycoplasma pneumoniae is the etiologic agent responsible for primary atypical pneumonia. Several species of mycoplasmas also appear to be involved in some cases of non-gonococcal urethritis and pelvic inflammatory disease.
Mycoplasmas have the ability to pass through 450-nm filters, as do chlamydiae, rickettsiae, and viruses. However, mycoplasma differ from these other small infectious agents in being able to grow, albeit slowly, on artificial media. Many mycoplasmas are initially mistaken for viruses. Their filterability is due not only to their small size but also the inherent flexibility of their cell envelope. Since mycoplasmas are only surrounded by a lipid membrane, they have been designated as the class Mollicutes (L. mollis and cutis, soft skin).
Variations in the size and shape of mycoplasmas can be ascribed in part to the lack of a cell wall, as can the variable staining obtained with Gram stains. Electron microscopy has shown that mycoplasmas have a variable and pleomorphic cellular morphology, even within a pure culture.
Some organisms assume a predominantly spherical appearance (300-800 nm in diameter) while others may form filaments of uniform diameter (100-300 nm), varying in length from 3 .mu.m to over 150 .mu.m. Thin sections reveal a simple ultrastructure consisting of a cell membrane and cytoplasm, including ribosomes and the characteristic nucleoid. There are no intracellular membranous structures.
Most mycoplasmas will form very small colonies (50 .mu.m-600 .mu.m in diameter), which can normally only be seen with a low power microscope. The classic "fried egg" appearance of typical mycoplasma colonies is due to an opaque, granular central zone of growth down into the agar and a translucent peripheral growth zone on the surface. However, not all mycoplasmas produce colonies with a "fried egg" morphology, especially primary isolates. Variations in colonial morphology are frequently dependent on the constituents and hydration of the growth medium.
Mycoplasmas usually divide, like other prokaryotes, by binary fission. In some instances genomic replication and cytoplasmic division are not precisely synchronized and become dissociated. A lag in cytoplasmic division yields multinucleated filaments, which subsequently form chains of coccoid cells and then fragment into individual cells.
Mycoplasmas have a circular genome of double-stranded DNA, one-fifth to one-half as large as that in most bacteria. This is evidently the smallest genome that can code for all the products needed for self-reproduction in an artificial medium.
Mycoplasmas have exacting nutritional requirements, especially for the lipids essential to synthesize their plasma membrane. The nutritional requirement of many mycoplasmas for exogenous sterol is unique among prokaryotes. It is usually met by animal serum, which contains cholesterol bound to a lipoprotein.
Mycoplasmas are difficult to identify and in the past were consistently confused with L-forms of eubacteria. The difficulty in detecting mycoplasma has implications both in microbiology (e.g., contaminated cell cultures) and in human and veterinary medicine (e.g., mycoplasma infection).
Although these obligate cellular parasites have exacting requirements for growth, mycoplasma contamination of cell cultures remains a great problem for many researchers.
These frequently undetected parasites are common infectants of cell cultures (Barile (1979) The Mycoplasmas, J. G. Tully and S. Razin, eds.: 425-474; McGarrity, et al. (1985) The Mycoplasmas, M. F. Barile and S. Razin, eds.: 353-390; Stanbridge (1971) Bacteriol 35: 206-227). The precise effect of the contaminant on cell structure can vary with the species or strain of mycoplasma, in addition to the cell type and growth medium. The unpredictable effects of mycoplasma contamination of cell cultures have included virtually every cell parameter, including changes in cytokine production, increased or decreased viral production and chromosome breakage.
Despite the documented nuclease activity of mycoplasmas (Neimark (1964) Nature 203: 549-550; Razin, et al. (1964) J. Gen. Microbiol. 36: 323-332) and their nutritional requirement for nucleic acid precursors (Razin (1962) J. Gen. Microbiol. 28: 243-250; Razin, et al. (1960) J. Gen. Microbiol. 22: 504-519), there is little information relating to the influence of mycoplasma infection on molecular biology techniques such as antisense translation inhibition.
In the antisense oligonucleotide approach to gene therapy, specific oligonucleotides are introduced into target cells. Following internalization, the oligonucleotides interfere in a sequence-specific manner with the RNA translation mechanism. Despite numerous reports of antisense effects in mammalian cells resulting from exogenous addition of oligonucleotides, fundamental questions about the stability and uptake of these compounds remain (Neckers, et al. (1992) CRC Crit. Rev. Oncogenesis 3: 175-231). Even less is known about the pharmacokinetics of these reagents in vivo Plainly, factors affecting these parameters must be controlled.
Others have attempted to devise ways of easily and quickly detecting mycoplasma contamination in cell cultures without much success. In one experiment, over 90% of [.sup.3 H]Uracil was found to be incorporated into mycoplasma RNA in a contaminated cell culture while only 10% was incorporated into the growing culture cells. However, .sup.3 H is difficult to detect by autoradiography or a geiger counter, and therefore provided a very poor (and slow) method for identifying mycoplasma contamination. Extended periods of autoradiography are necessary to visualize the incorporation of [.sup.3 H]Uracil into RNA.
Tritium labeled thymidine ([.sup.3 H]T) and uridine ([.sup.3 H]U) have also been used in a bioassay based upon the degradation of ([.sup.3 H]T) by a cell free culture supernatant (Merkenschlager (1988) Immunology 63(1): 125-131). In this method, mycoplasma contamination was detected by pyrimidine phosphorylase activity on the labeled nucleotides.
Other experimenters have also used various tritium labeled nucleosides and found that mycoplasma RNA is tritiated by these compounds more efficiently than mammalian cells (Schneider (1974) Experimental Cell Research 84(1-2): 311-318; McIvor (1978) Journal of Bacteriology 135: 483-489). Although the uptake and incorporation of tritium-labeled nucleosides into mycoplasma RNA has been used to determine the extent of cell culture contamination, tritium's weak radioactivity has made rapid diagnosis difficult. Experiments involving stronger radioactive isotopes, such as .sup.32 P, were unsuccessful since the labeled nucleotides were inefficiently incorporated into the mycoplasma RNA (Hellung-Larsen (1976) Experimental Cell Research 99(2): 295-300).
An alternative method of detecting mycoplasma contamination in a cell culture involves using specific labeled probes that only hybridize with mycoplasma gene sequences (Gobel (1987) Israel Journal of Medical Sciences 23: 735-741). This procedure, however, also requires substantial preparation time to isolate the cell culture DNA, prepare the probe, and perform the hybridization assay.
It would be desirable to have a method which incorporated the ease of the metabolic techniques (e.g.: tritium experiments) with a stronger means of preferentially labeling the mycoplasma. Unfortunately, mycoplasma do not seem to take up the .sup.32 P labeled nucleotides at the fast rate seen with the tritium labeled nucleotides. The preferred embodiment of the present invention overcomes this problem by providing a method of quickly identifying mycoplasma contamination using high specific activity isotopes such as .sup.32 P and .sup.35 S.